Recipient Organization
BioProtection Systems Corporation
2901 South Loop Drive Suite 3360
Ames,IA 50010
Performing Department
(N/A)
Non Technical Summary
This application addresses the USDA NIFA "Global Food Security and Hunger" primary societal challenge area by developing a countermeasure against a potential agricultural disease threat - Rift Valley fever virus (RVFV). RVFV is an arthropod-borne pathogen that often results in severe morbidity and mortality in both humans and livestock. RVF manifests itself in the vast majority of individuals that become infected. In fact, unlike a West Nile virus (WNV) infection, which has no clinical manifestation in 80% of infected individuals, approximately 90% of humans infected with RVF virus show clinical signs of the disease. While infections in humans are typically mild and present as self-limiting febrile illnesses, RVFV infections can progress to more severe disease including fulminant hepatitis, encephalitis, retinitis, blindness, or a hemorrhagic syndrome in approximately 2% of affected individuals. However, statistics from recent outbreaks suggest that the case fatality rate from RVFV infection is significantly increasing (up to 45%) in naive populations. Human RVFV infections are usually preceded by transmission from wild to domestic animal hosts, recognized by sudden and devastating impact on livestock. In sheep, mortality in lambs under 2 weeks of age approaches 100%, reaches 30% in older animals and abortions approach 100%. Cattle also show high abortion rates (up to 100%) with adult mortality averaging 10%. As RVFV's geographic range continues to spread, it presents a real threat to naive populations around the world by accidental introduction or a bioterror/agroterror event. The lack of prophylactic and therapeutic measures, the potential for human-to-human and animal-to-animal transmissions, and the significant threat to livestock associated with RVFV make infection with this pathogen a serious public health concern. RVFV epizootics and epidemics might rapidly overwhelm the capacities of the public health and veterinary medical communities to provide rapid diagnostic testing, distribution of countermeasures and adequate medical care. The development of safe and efficacious RVFV vaccines has proven to be quite difficult. Live-attenuated countermeasures have a high potential to revert to the deadly wildtype virus, while subunit candidates usually require several vaccination/boost regimens to initiate immunity. In addition, most of the tested live attenuated vaccine candidates do not follow the DIVA concept which makes it impossible to differentiate infected from vaccinated animals - a crucial feature in outbreak situations. The use of VLPs is a promising alternative approach for the development of a safe and efficient RVFV vaccine. RVF VLPs exhibit morphology similar to that of wild-type viruses and have wild type tropism with comparable cellular uptake and intracellular trafficking. VLPs present viral antigens in a native conformation allowing for effective recognized by the immune system. The VLP-based vaccine candidates against RVFV described in this proposal are safe, show high efficacy in two rodent models, and are therefore a promising concept to combat emerging RVFV.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Goals / Objectives
The proposed project addresses a key gap in vaccine technology to protect agricultural health: the need for a safe and efficacious Rift Valley fever virus (RVFV) countermeasure. The developed vaccine technology will satisfy very important unmet national, societal and commercial needs. We will establish a RVF virus-like particle (VLP) production methodology that allows the production of a RVFV vaccine candidate for all proposed studies. Development and optimization of production methods including VLP purification and concentration to increase overall vaccine yield (sufficient for small scale livestock studies) will be established. A comparison between infected and vaccinated animals will confirm that the proposed RVFV countermeasure follows the DIVA (to differentiate infected and vaccinated animals) concept. Furthermore, efficacy and initial safety studies in the natural disease host, sheep, will demonstrate vaccine efficacy and safety in livestock Objective 1: Characterize and Optimize a Rift Valley Fever Virus-like Particle Production System: We will develop a scalable production system for RVF VLPs using an insect cell system, and focus on RVF VLP purification for the generation of highly concentrated, purified material in sufficient quantities to perform the proposed animal studies. Recombinant baculovirus-derived VLPs are an attractive and cost-effective approach for the generation of RVF VLPs because of the potential for high yield production of VLPs in insect cells. Objective 2: Verify DIVA Properties of RVF VLP-based Vaccine Candidate: We will show that RVF VLPs adhere to the DIVA concept. Because RVFV DNA and other gene products (e.g., polymerase, NSs, NSm) are omitted during the generation of RVF VLPs, the ability to identify viral genes by RT PCR and the strong immune response generated against the NSs protein in infected animals will allow for the distinction between infected and RVF VLP-vaccinated animals. Objective 3: Demonstrate Vaccine Efficacy in Sheep: We will evaluate the protective efficacy and safety of the RVF VLP-based vaccine candidate in sheep (an established animal model and natural host for RVFV). We will monitor survival and abortion rates as a response to a lethal challenge with infectious RVFV. We will further examine vaccine efficacy and safety by measuring challenge virus titer in serum and organ samples, analysis of liver enzymes and hematology, RVFV-specific IgG, neutralizing antibody levels and cytokine analysis and pathology and virus replication via immunohistochemistry. The analysis of liver enzymes, hematology and the study of immune correlates involve proven methodologies and assays on hand.
Project Methods
VLP production and characterization: Current lab scale VLP generation methodology is insufficient for the preparation of RVF VLPs in the quantity required for studies in livestock. An insect cell system will be used and developed to a scalable production system for RVF VLPs. This system will be used for the generation of highly concentrated and purified material in sufficient quantities to perform the proposed animal studies. Standard centrifugation protocols will be employed to eliminate suspension cells/cell debris and concentrate/purify VLPs to levels suitable for proposed animal studies. Tangential Flow filtration and depth filtration will be employed to further concentrate and purify VLPs. Polyethylene glycol precipitation will be performed as an alternative method for the concentration and purification of clarified RVF VLPs. Further downstream purification steps will include chromatography (e.g., size exclusion, mixed mode ion exchange and polymethcrylate monoliths. Purity, integrity and recovery of the VLPs will be evaluated for (1) total protein in a BCA assay, by (2) Western blotting and Ag-specific ELISAs for RVFV GN/GC and N expression and (3) for the ability of VLPs to bind to permissive cells. A FACS-based method will be used to measure surface glycoprotein-dependent binding of RVF VLPs to permissive cells as an indication of the RVFV G structural integrity. RVF VLP morphology will be evaluated by transmission electron microscopy (TEM). The EM findings in combination with the total protein analysis values will allow to visualize the quality and estimate the quantity of VLPs/ml that were generated. VLP DIVA Properties: We will demonstrate that RVF VLPs adhere to the DIVA concept. Because, with the exception of GN and GC and N, all RVFV genes and most gene products are omitted during the generation of RVF VLPs, the ability to identify viral genes by RT PCR and a strong immune response against the NSs proteins in infected animals, and the lack thereof in VLP-vaccinated animals will be used for differentiation of infected or vaccinated animals. To demonstrate that the RVF VLPs adhere to the DIVA concept mice will be infected with either RVFV MP12 virus or vaccinated with the RVF VLPs. 28 days p.i. or vaccination, serum and organ samples will be harvested and analyzed for the presence of antibodies specific for RVFV GC, GN, N and NSs and RVFV genetic material. An alternative strategy will focus on the lack of genetic material in the RVF VLP vaccine. Using quantitative (q) real-time RT-PCR from serum and/or organ samples (e.g., liver) from MP12-infected and VLP-vaccinated mice, we will demonstrate that RVFV genetic material is detectable in infected animals, but not vaccinated animals. VLP Vaccine Efficacy: We will examine VLP vaccine efficacy and safety in sheep by measuring challenge virus titer in serum and organ samples, analysis of liver enzymes and hematology, RVFV-specific IgG, neutralizing antibody levels and cytokine analysis and pathology and virus replication via immunohistochemistry. The analysis of liver enzymes, hematology and the study of immune correlates involves proven methodology and assays on hand.